The Polo-box domain (PBD) is critical for mitotic functions of PLK1 and has shown to be required for substrate recruitment and sub-cellular localization. The McInnes laboratory has made significant progress toward the development of PLK1 selective inhibitors based on PBD inhibition that have minimal activity on the PLK3 tumor suppressor and which retain activity against PLK1 site mutants that are resistant to clinically utilized ATP competitive inhibitors. These compounds will be high value chemical biology probes for further establishing the phenotypic consequences of inhibiting PLK1 in a non-ATP competitive fashion through the PBD, in validating the PBD as an anti-mitotic drug target and in their potential as next generation therapeutics. This proposal is significant and innovative with respect to the following: 1) An innovative strategy called REPLACE has been developed to generate cell cycle specific CDK inhibitors as anti-tumor therapeutics. Furthermore the McInnes laboratory validated this strategy for the PLK1 PBD revealing that fragment ligated peptidic inhibitors discovered though REPLACE have respectable anti-proliferative activity and phenotypes consistent with target inhibition. 2) Chemical biology probes that target the PBD will be highly selective for PLK1 and possess minimal affinity for the PLK3 tumor suppressor. This is important to further establish the phenotypic consequences of blocking the PLK1 PBD selectively and also to avoid deleterious effects of PLK3 inhibition in the development of anti-tumor therapeutics. We have already demonstrated that our most potent PLK1 PBD domain inhibitors have minimal activity against PLK3. 3) PBD targeted compounds will retain antitumor activity against tumor cells that have acquired resistance to ATP-based inhibitors. As described above, a single point mutant (C67V) is capable of rendering PLK1 resistant to structurally diverse ATP-based inhibitors. Targeting non-catalytic functions of PLK1 should result in less selective pressure for resistance and PBD inhibitors might be used in combination with ATP-based inhibitors as a synergistic means of PLK1 targeting in the clinic. 4) Current lead compounds induce a phenotype in treated cells that recapitulates a PLK1 knockdown. This stands in contrast to weakly binding small molecule PBD inhibitors reported that only induced partial effects, and may not possess a desirable level of anti-tumor activity. Some of our observed phenotypes are novel and suggestive of anaphase catastrophe, known to promote tumor selectivity. 5) Novel PBD inhibitors will be tumor selective compounds. Recent discoveries that PLK1 inhibition is synthetically lethal in PTEN deficient, mutant Ras and p53 deficient cancers will be exploited to generate tumor selective compounds. Non-peptidic inhibitors will be used in cellular studies to confirm tumor cell specificity, PLK1 selectivity, and cellular phenotypes consistent with on target inhibition.